The present invention relates generally to plant growth and development and more specifically to methods of modifying brassinolide activity in plants by modulating cytochrome P450 activity.
A plant is considered healthy when it can carry out its physiological functions, such as cell division, differentiation, development, photosynthesis, absorption and translocation of water and nutrients from the soil, metabolism, reproduction, and storage of food supplies, without disruption. When plant functions are disturbed by pathogens or insects, the plants become diseased or destroyed. Disease can be defined as the malfunctioning of plant host cells and tissues caused by continuous irritation by a pathogenic agent or insect. A disease involves abnormal changes in the form, physiology, or behavior of the plant.
Representative insects or pests that attack plants include Coleoptera and Lepidoptera such as western corn root worm (Diabrotica virgifera virgifera), northern corn root worm (Diabrotica longicornis barberi), southern corn rootworm (Diabrotica undecimpunctata howardi), cotton bollworm, European corn borer, corn root webworm, pink bollworm and tobacco budworrn. The transgenic plants are preferably monocotyledoneous or dicotyledoneous plants. Plant pathogenic bacteria also cause a variety of plant disease symptoms. About 80 species of bacteria (e.g., Pseudomonas viridiflava, Xanthomonas campestris pv. asclepiadas, Xyella fastidiosa, Acidovorax albilineans, and Acidovorax avenae sspl citrulli) cause disease in plants, including fruit rot, galls, wilts, blight, and leaf spots. As bacteria multiply quickly, controlling them early in the disease process is critical. Copper and streptomycin compounds are the only chemical compounds currently available for the control of bacterial diseases.
Genetic engineering of plants, which entails the isolation and manipulation of genetic material, e.g., DNA or RNA, and the subsequent introduction of that material into a plant or plant cells, has changed plant breeding and agriculture considerably over recent years. Increased crop food values, higher yields, feed value, reduced production costs, pest resistance, stress tolerance, drought resistance, the production of pharmaceuticals, chemicals and biological molecules as well as other beneficial traits are all potentially achievable through genetic engineering techniques. Genetic engineering techniques supplying the genes involved in pathogen resistance have the potential to substantially affect crop production.
Traditionally, the control of plant stature has been through the process of selective breeding. Often dwarf plants are chosen for their ornamental value or their improved ability to survive under mechanical stress, such as high wind. However, this breeding process can take many years. An alternative way to rapidly create dwarf plants is by the exogenous application of certain organic compounds, such as the gibberellin biosynthesis inhibitor, uniconazole. However, these compounds are expensive and must be applied throughout the plant life cycle.
Light also has an important role in plant development, both for photosynthesis and as a developmental cue. A variety of photoreceptors respond to the quality, quantity, direction and duration of the light environment. In Arabidopsis there are five red/far-red absorbing phytochromes (phyA-phyE), two blue/UVA absorbing cryptochromes (cry1 and 2) and the less understood UVB photoreceptors. All affect gross morphological changes in seedling development as they deetiolate, making the transition from growth in the dark to growth in the light (C. Fankhauser and J. Chory, Annu Rev Cell Dev Biol 13:203-29, 1997). Genetic analysis demonstrates a complex web of interactions between these photoreceptor signaling pathways (Casal and Mazzella, Plant Physiol 118:19-25, 1998; M. M. Neff and J. Chory, Plant Physiol. 118:27-35, 1998; L. Hennig et al., Planta 208:257-263, 1999; G. Lasceve et al., Plant Physiol 120:605-614, 1999). There is also a distinct class of photoreceptors, the phototropins (e.g. NPH1), which effect the directional growth of seedlings (E. Liscum and W. R. Briggs, Plant Cell 7:473-485, 1995; E. Huala et al., Science 278:2120-2123, 1997; J. M. Christie et al., Science 282:1698-701, 1998), a process that can be modified by the activity of phytochromes (B. M. Parks et al., Plant Physiol. 110:155-162, 1996).
Plant hormones can also contribute to photomorphogenic responses. Some photomorphogenetic mutants resemble mutants involved in phytohormone biosynthesis or sensing. For example, the GA signaling mutant spindly resembles plants with mutations in phyB, which have long stems, pale leaves and early flowering. This phenotype can also be mimicked in wild type plants by the application of GA3 (S. E. Jacobsen and N. E. Olszewski, Plant Cell 5:887-896, 1993). Genetic analysis of GA and phytochrome mutants points to interactions between these two signal transduction systems for certain responses (J.Chory, Plant Cell 9:1225-34, 1997). However, other responses, such as flowering, are likely to be independently controlled by both systems (M. A. Blxc3xa1zquez and D. Weigel, Plant Phys 120:1025-32, 1999).
Gibberellins are not the only hormones that are involved in light signaling. Auxins clearly have some role in photomorphogenesis. For example, auxin transport is affected in a light dependent manner (P. J. Jensen et al., Plant Physiol 116:455-462, 1998). Genetic analysis also points towards a role for auxin in light signal transduction. The shy2 mutation, identified in a suppressor screen for mutants with reduced levels of all phytochromes (Kim et al.[In Process Citation]. Plant J 15:61-68) or in a null mutant of phyB (J. W. Reed et al., Genetics 148:1295-1310, 1998), resides in the auxin induced gene IAA3 (Q. Tian and J. W. Reed, Development 126:711-21, 1999). A third example of the interplay between photomorphogenesis and phytohormones is that many brassinosteroid mutants have been identified in genetic screens for plants that can undergo deetiolation in the absence of a light cue (for review see (J. Li and J. Chory, Exp Bot 50:275-282, 1999)). When these mutants are grown in the dark, their seedlings have short hypocotyls with cotyledons that begin to develop as if growing in the light. As adults, these mutants are dwarfs with dark-green, epinastic leaves and short stems and petioles. They are slow growing with delayed senescence. Each of these adult phenotypes is essentially the opposite of mutants lacking phytochrome B (for review see (Chory, 1997, supra)).
Genetic screens for loss-of-function mutations have led to the identification of many loci thought to be involved in photomorphogenesis (Fankhauser and Chory, supra). However, these screens may miss important components of light signal transduction that are either redundant members of a gene family or are essential for survival. The role of such genes may only be identified in gain-of-function mutant screens. One method for targeting gain-of-function mutations is through extragenic suppressor analysis (G. Prelich, Trends Genet 15:261-6, 1999).
This approach has been used successfully in Arabidopsis to identify dominant or semidominant mutants involved in light signal transduction (A. Pepper and J. Chory, Genetics 145:1125-37, 1997; Kim et al., supra; Reed et al., 1998, supra). However, positional cloning of dominant or semidominant extragenic suppressors can be difficult and time consuming if they do not have a phenotype in a wild type background. shy2 is the only dominant, extragenic suppressor mutation cloned by map based methods in Arabidopsis and has a striking phenotype in a wild type background (Tian and Reed, supra).
A gene tagging approach has been used to circumvent the difficulties of map-based cloning of mutations in Arabidopsis. In this approach, mutants are generated by transformation with Agrobacterium mediated transfer-DNA (T-DNA). Since the T-DNA sequence is known, mutations that are tagged by the transgene can be easily identified and cloned (F. J. Behringer and J. I. Medford, Plant Molecular Biology Reporter 10:190-198, 1992; Y.-G. Liu et al., Plant J 8:457-463, 1995.
However, these mutations are primarily caused by the loss of gene function. Thus, the amount of information that can be gleaned from their identification is limited. A modification of T-DNA tagging has been developed that specifically targets gain-of-function mutations. In this approach, multimerized copies of enhancer elements from the cauliflower mosaic virus (CaMV) 35S promoter are incorporated near the right border of a T-DNA. When these enhancers are inserted near a gene, its transcription may be enhanced; resulting in a dominant, tagged mutation (R. Walden et al., Plant Mol Biol 26:1521-8, 1994).
The present invention is based on the discovery that bas1 gene in Arabidopsis thalia encodes a cytochrome P450 (CYP72B1), which has a role in brassinosteroid signaling or synthesis. Overexpression of the bas1 gene in plants causes a dark green, dwarf phenotype which mimics plants that have low levels of the plant hormone, brassinolide. Overexpression of the bas1 gene also increases resistance to insects in plants. Biochemical analysis shows that CYP72B1 is a C-26 hydroxylase of brassinolide, targeting it for inactivation. Transgenic plants engineered to overexpress bas1 gene have severely reduced levels of brassinolide and some brassinosteroid precursors. Overexpression of the bas1 gene in Tobacco (Nicotiana tabacum) confers a similar phenotype, indicating that this gene is functionally active in a divergent plant family. Based on these observations, the present invention provides plants genetically modified with bas1 gene, or functional homologues or functional fragments thereof, wherein such plants exhibit modulated brassinosteroid signaling or synthesis as compared to wild-type plants.
Accordingly, in a first embodiment, the invention provides a BAS1 polypeptide that modulates brassinolide activity in plants. Also included are polynucleotides encoding BAS1 and antibodies that bind to BAS1.
In another embodiment, the invention provides a genetically modified plant comprising at least one exogenous nucleic acid sequence encoding a cytochrome P450, e.g., BAS1 polypeptide or functional equivalent, in its genome or at least one regulatory sequence that modifies expression of endogenous cytochrome P450, e.g., bas1 gene and which is characterized as having modulated brassinolide activity compared to a wild-type plant. Such genetically modified plants may exhibit dwarfism with dark-green leaves in adult plants.
In another embodiment, the invention provides a method for genetically modifying a plant cell such that a plant, produced from the cell, is characterized as having modulated brassinolide activity as compared with a wild-type plant. The invention method for modifying a plant cell comprises introducing at least one exogenous cytochrome P450, e.g., bas1 polynucleotide, into a plant cell to obtain a transformed plant cell, and growing the transformed plant cell under conditions that permit expression of cytochrome P450, e.g., bas1 gene product, thereby producing a plant having modulated brassinolide activity.
In another embodiment, the invention provides a method of producing a genetically modified plant characterized as having dwarf adult stature with dark green foliage, by contacting a plant cell with a vector containing an exogenous nucleic acid sequence comprising at least one structural gene encoding a cytochrome P450, e.g., BAS1 polypeptide, the gene being operably associated with a regulatory sequence that causes overexpression of the gene, to obtain a transformed plant cell, producing a plant from the transformed plant cell; and selecting a plant exhibiting dwarf adult stature with dark-green foliage.
In another embodiment, the invention provides a genetically modified plant having a transgene increasing expression of bas1 gene, chromosomally integrated into the genome of the plant.
In another embodiment, the invention provides a method of producing a genetically modified plant characterized as being hyperresponsive to brassinolide, by contacting a plant cell with a vector containing an exogenous nucleic acid sequence encoding a product that disrupts or interferes with expression of cytochrome P450, e.g., BAS1 polypeptide, which nucleic acid is operably associated with a promoter, to obtain a transformed plant cell, producing a plant from the transformed plant cell; and selecting a plant exhibiting hyperresponsiveness to brassinolide in a light-dependent manner.
In another embodiment, the invention provides a method for genetically modifying a plant cell such that a plant, produced from the cell, is characterized as having modulated ecdysteroid activity as compared with a wild-type plant. In this embodiment, the invention method comprises introducing at least one exogenous cytochrome P450, e.g., bas1 polynucleotide, into a plant cell to obtain a transformed plant cell; and growing the transformed plant cell under conditions the permit expression of the cytochrome P450, e.g., bas1 gene product, thereby producing a plant having modulated ecdysteroid activity.
In another embodiment, the invention provides a method of producing a genetically modified plant characterized as having increased disease or insect resistance as compared to a corresponding wild-type plant. The method includes contacting plant cells with nucleic acid encoding an BAS1 polypeptide or functional fragment thereof, operatively associated with an expression control sequence, to obtain transformed plant cells; producing plants from the transformed plant cells; and selecting a plant exhibiting increased disease or insect resistance. A method for genetically modifying a plant cell such that a plant produced from the cell is characterized as having increased disease or insect as compared with a wild-type plant is also provided. The method includes introducing an isolated polynucleotide encoding a BAS1 polypeptide into a plant cell to obtain a transformed plant cell, and growing the transformed plant cell under conditions which permit expression of BAS1 polynucleotide thereby producing a plant having increased disease or resistance.
In a further embodiment, a method is provided for producing a genetically modified plant characterized as having increased disease or insect resistance as compared to the corresponding wild type plant by contacting a susceptible plant with a BAS1 promoter-inducing amount of an agent necessary to elevate BAS1 gene expression above BAS1 expression in a plant not contacted with the agent. For example, the agent may be a transcription factor or a chemical agent, such as dexamethasone (DEX).
A method is also provided for producing genetically transformed, disease or insect resistant plants, by introducing into the genome of a plant cell, to obtain a transformed plant cell, a nucleic acid sequence having an expression control sequence operably linked to a polynucleotide encoding a BAS1 polypeptide. The invention also provides plants, plant tissue, and seeds produced by plants produced by the methods of the invention.
In yet another embodiment, a method is provided for identifying novel disease or insect resistance genes or genes capable of inducing reduced stature in plants by probing a nucleic acid library with at least a fragment of a polynucleotide encoding BAS1, and selecting those clones that hybridize with the fragment.
In another embodiment, the invention provides an isolated nucleic acid sequence comprising a non-coding regulatory sequence isolated upstream from a bas 1 gene, wherein said nucleic acid sequence contains at least one restriction site for cloning a heterologous nucleic acid sequence of interest. An exemplary bas1 regulatory sequence is shown in FIG. 1C (SEQ ID NO:16). The invention also provides a nucleic acid construct comprising, reading from the 5xe2x80x2 to 3xe2x80x2 direction, a 5xe2x80x2 non-coding sequence isolated from bas1 gene and a nucleic acid sequence encoding a structural gene, wherein said nucleic acid sequence is heterologous to said 5xe2x80x2 non-coding sequence. The construct is useful for the production of transgenic plants which express a gene of interest in a tissue-specific manner, for example.